Mycol. Res. 107 (7): 822–830 (July 2003). f The British Mycological Society
822
DOI: 10.1017/S0953756203008050 Printed in the United Kingdom.
Morphology of Verticillium dahliae and V. tricorpus on
semi-selective media used for the detection of V. dahliae in soil
Jan-Kees C. GOUD1, Aad J. TERMORSHUIZEN1 and Walter GAMS2
1
Biological Farming Systems Group, Wageningen University, Marijkeweg 22, 6709 PG Wageningen, The Netherlands.
Centraalbureau voor Schimmelcultures, Postbus 85167, 3508 AD Utrecht, The Netherlands.
E-mail : [email protected]
2
Received 27 January 2003; accepted 8 May 2003.
The morphology of two soil-borne Verticillium species, V. dahliae and V. tricorpus, was studied on two semi-selective
agar media, in the absence and presence of soil. Morphology of the fungi differed considerably between the media, with
respect to presence and shape of microsclerotia, dark hyphae (i.e. short melanised hyphae attached to the microsclerotia)
and dark mycelium (i.e. melanised mycelium throughout the colony). On modified soil extract agar (MSEA), a pectate
based agar, V. dahliae always had globose to elongate microsclerotia, without dark hyphae or dark mycelium, whereas
V. tricorpus always had dark hyphae or dark mycelium, and microsclerotia, whenever present, were globose to irregular
in shape. On ethanol agar (EA), V. dahliae had large microsclerotia and abundant dark hyphae, whereas V. tricorpus did
not form microsclerotia, but always abundant dark mycelium. For the first time we observed the formation of dark
hyphae by V. dahliae to a great extent. In the presence of soil, most characteristics were less pronounced, and V. dahliae
microsclerotia were smaller, but V. tricorpus produced large microsclerotia, even when they were absent in pure culture.
Morphological characteristics suitable for discrimination between the two species on MSEA plates in the presence of soil
were selected and tested with fresh isolates from agricultural fields. The two fungi could be distinguished using qualitative
characteristics and microsclerotial size. Molecular analysis and morphology on potato dextrose agar confirmed all
identifications made on soil dilution plates.
INTRODUCTION
Representatives of the form-genus Verticillium Nees
1816 in the strict sense (anamorphs of the Phyllachorales (Messner et al. 1996), are commonly found in
agricultural soils (Domsch, Gams & Anderson 1980).
The genus includes the virulent plant pathogenic
species V. dahliae Kleb. 1913, V. longisporum (Stark)
Karapapa, Bainbridge & Heale 1997 and V. albo-atrum
Reinke & Berth. 1879, which have low saprotrophic
abilities, V. nubilum Pethybr. 1918 and V. nigrescens
Pethybr. 1919 which are saprobes and weak pathogens,
and V. tricorpus Isaac 1953 which has intermediate
saprotrophic ability and is pathogenic to a limited
number of crops. All these species can be associated with
the same crop, e.g. potato or tomato (e.g. Isaac 1967,
Skotland 1971, Domsch et al. 1980, Pegg & Brady
2002).
The six species can be differentiated morphologically
by the types of resting structures they form in and on
the surface of plant material, and on many artificial
agar media, such as potato dextrose agar (PDA). V.
dahliae and V. longisporum form microsclerotia, which
are black melanised clumps, formed by budding of
mycelial cells ; V. albo-atrum forms melanised resting
mycelium ; V. nigrescens and V. nubilum form chlamydospores, and V. tricorpus forms microsclerotia, resting
mycelium and chlamydospores. The morphological
differences of the microsclerotia of V. dahliae and
V. tricorpus are pronounced on PDA, as described by
Isaac (1949, 1953) and Smith (1965) : V. tricorpus forms
large and irregularly shaped microsclerotia, usually
with melanised hyphae growing from them, and basally
pigmented conidiophores, whereas V. dahliae forms
smaller and oval to elongate microsclerotia which are
sharply differentiated from the hyaline mycelium and
hyaline conidiophores. Moreover, V. tricorpus often
causes a yellow discoloration of the PDA medium
upon first isolation. V. longisporum is known to be a
heterodiploid between V. dahliae and V. albo-atrum.
Morphologically V. longisporum is most similar to
V. dahliae but it can be differentiated from V. dahliae in
pure culture by the shape of the microsclerotia, the
number of phialides per node and, particularly, its
larger conidia, 8r2.5 mm. V. longisporum is mainly
known from cruciferous hosts (Karapapa et al. 1997).
J. C. Goud, A. J. Termorshuizen and W. Gams
823
Table 1. Origin of isolates of Verticillium dahliae and V. tricorpus used in Experiment 1.
Species
Isolatea
Host of origin
Year
of isolation Location
V. dahliae
A59 mc-1
Blackberry (Rubus fruticosus)
1996
V. dahliae
A45 mc-1
Strawberry (Fragaria sp.)
1996
V. dahliae
A60 mc-1
Maple (Acer sp.)
1993
V. dahliae
A56 mc-1
Rose (Rosa sp.)
1996
V. dahliae
A63 mc-1
Forsythia sp.
1997
Alstroemeria sp.
1996
V. tricorpus A36 mc-1
V. tricorpus 127.79A mc-1 Tomato (Lycopersicon esculentum) 1979
V. tricorpus 384.84 mc-1 Potato (Solanum tuberosum)
1984
V. tricorpus 227.84 mc-1
Potato (Solanum tuberosum)
1984
V. tricorpus Vtx mc-1
–
–
a
b
Isolation by
Spijk, The Netherlands
J. W. Veenbaas-Rijks, Plant
Protection Service
Elst, The Netherlands
J. W. Veenbaas-Rijks, Plant
Protection Service
Swolgen, The Netherlands
Auf ‘m Keller, Plant
Protection Service
Elst, The Netherlands
J. W. Veenbaas-Rijks, Plant
Protection Service
Aalsmeer, The Netherlands
B. Wessels, Plant Protection
Service
Rijnsburg, The Netherlands
J. W. Veenbaas-Rijks, Plant
Protection Service
Roxborough, New Zealand
B. Taylorb
Wageningen, The Netherlands J. van der Spek, Institute
for Plant Protection Research
Dronten, The Netherlands
T. Hofman,
Wageningen University
–
–
Voucher cultures are preserved in CBS (Utrecht).
Unknown.
Because of the resemblance of V. dahliae and V. tricorpus on poor isolation media, our study is mainly
concerned with these two species.
In a number of field crops, but also many woody
species, V. dahliae can cause serious wilt disease,
whereas V. tricorpus is generally harmless (Hiemstra
1998). V. tricorpus has even been recommended for the
biocontrol of Rhizoctonia solani in cotton seedlings
(DeVay et al. 1988, Paplomatas, Tzalavaras & DeVay
2000) and V. dahliae in potato (Davis et al. 2000).
There is a need for the reliable assessment of soil population densities of V. dahliae in order to predict possible
problems in tree nurseries and agricultural crops. Plating soil samples on semi-selective media is currently
the most reliable way to assess population densities of
V. dahliae in soil (Termorshuizen et al. 1998). Microsclerotia are detected indirectly by giving rise to
characteristic new colonies with microsclerotia on a
semi-selective agar medium. However, microsclerotia
of V. tricorpus, when present in these soil samples, can
also form similar colonies with microsclerotia. Rich
media, like PDA, cannot be used during quantification
of V. dahliae in soil, because rapidly growing and
sporulating fungi, present in all soils, will overgrow
V. dahliae, resulting in failure of quantification. While
morphological differences are pronounced on rich
media, on (poor) semi-selective media V. dahliae and
V. tricorpus are easily confused, even by experienced
researchers (Termorshuizen et al. 1998). Isolated
colonies can be characterised using molecular techniques (Robb et al. 1994). However, the method of
transferring colonies from semi-selective media to
PDA, and purification for identification of species is
too laborious to be employed as a standard procedure.
Therefore it is important for disease prediction to
compare the morphology of the two species on the
original semi-selective media directly, and describe
differential characteristics of the two species.
In this study we : (1) compared the morphology of
V. dahliae and V. tricorpus on semi-selective media in
pure culture and in the presence of insterile soil ; (2)
distinguish characteristics that can be used to separate
the two species on semi-selective media in the presence
of soil ; and (3) test these characteristics on colonies
from soil samples of agricultural fields. The identity of
isolates was always verified by molecular analysis.
MATERIALS AND METHODS
In Experiment 1, five single-spore isolates of both
Verticillium dahliae and V. tricorpus were studied. In
Table 1 these isolates and their origin are listed. Morphology was first studied in pure culture on PDA to
determine the species : isolates with irregularly shaped
microsclerotia, chlamydospores, melanised mycelium,
and yellow discoloration of the medium were classified
as V. tricorpus. Isolates with globose, oval to elongate
microsclerotia and without chlamydospores or melanised mycelium were classified as V. dahliae (Isaac
1949, 1953, Smith 1965). All isolates fitted these
descriptions, except for the V. tricorpus isolates 384.84
mc-1 and Vtx mc-1 (Table 1) which did not form
microsclerotia on PDA, and did not show yellow
discoloration of the medium.
As experimental treatment, two semi-selective media
were compared : modified soil extract agar (MSEA)
(Harris, Yang & Ridout 1993), which is a pectate-based
medium often used for quantitative detection in soil,
and ethanol agar medium (EA) (Nadakavukaren &
Horner 1959), often used for isolating V. dahliae
from plant material. Each semi-selective medium
was amended with 50 mg lx1 oxytetracycline after
Distinction of Verticillium dahliae and V. tricorpus
autoclaving as the single bacteriostatic agent. Media
for Experiment 1 were produced once, to avoid batch
to batch variation. Ten microsclerotia per plate, produced on PDA covered with cellophane to stimulate
formation of microsclerotia (DeVay et al. 1974), were
placed individually on the two media.
Colony growth of Verticillium species was studied
with and without the presence of soil. For the latter
treatment, 0.8 ml a suspension of non-sterile, Verticillium-free sandy soil was added to the surface of the
inoculated agar plates. The soil had been steamed and
cropped with wheat for 6 wk before the start of the
experiment. During the experiment, the soil was treated
in the same way as a soil sample is treated during
the protocol for quantitative detection of V. dahliae
(Harris et al. 1993) : 12.5 g of dry soil was sieved, the
20–106 mm fraction was suspended in 0.08 % water
agar, and 0.8 ml of this suspension was spread over the
agar surface in a 9.4 cm diam. Petri dish, after which
the plates were air dried for 15 min. The inoculated
Petri dishes, with or without soil, were incubated upside
down in closed plastic bags for 6 wk at 20 xC. Five Petri
dishes were incubated per treatment per isolate with
and without soil.
Numbers of microsclerotia and their shape were
recorded for each colony. Length and width of ten
microsclerotia per colony were measured on three
colonies of each combination. The microsclerotia to be
measured were randomly chosen from the area halfway
between the centre and the edge of the colony. Presence
and intensity of dark melanised mycelium, growing
radially from the centre throughout the colony was
recorded (Figs 2, 6). The intensity was estimated as
the percentage of the colony covered by this type of
mycelium. In our study, this type of mycelium will be
referred to as ‘dark mycelium ’. The term ‘dark hyphae’
will be used for short portions (shorter than 120 mm) of
melanised hyphae. Dark hyphae usually arise from
microsclerotia (Figs 4, 7), but they can also occur singly
or several together without connection to a microsclerotium. Dark hyphae cannot be observed properly
when dark mycelium is abundant. Colony characteristics like the pattern in which microsclerotia are
formed, radial (Fig. 1), scattered (Figs 5–6), or intermediate, and yellow discoloration of the agar medium,
were recorded. Chlamydospores were not scored because they were regarded unsuitable for discrimination,
for two reasons : (1) chlamydospores are mostly hyaline
and therefore difficult to spot under a dissecting microscope ; and (2) similar chlamydospores can be formed
by various soil-borne fungi and therefore usually occur
abundantly throughout semi-selective soil plates. Size
of individual microsclerotial cells was not included in
the study, because : (1) individual cells cannot be observed properly through a dissecting microscope ; and
(2) preliminary observations revealed overlap between
cell sizes of V. dahliae and V. tricorpus.
In Experiment 2, ten individual colonies on MSEA
soil plates surface-inoculated with a 0.8 ml soil
824
suspension from agricultural fields were examined.
Only colonies with microsclerotia were taken into account. Mainly small colonies were chosen, with fewer
than 25 microsclerotia, as they are the most difficult to
classify as either V. dahliae or V. tricorpus. Length and
width of ten microsclerotia were measured, the pattern
in which the microsclerotia were formed, and the presence or absence of connected dark hyphae or dark
mycelium was scored. Based on the morphological
characteristics of Experiment 1, isolates of Experiment
2 were classified as V. dahliae or V. tricorpus. Then,
pure isolates of these colonies were obtained by retrieving individual microsclerotia from these colonies,
washing them several times in sterile demineralised
water, and re-plating them on MSEA. Colonies were
transferred to clean MSEA plates until they were pure,
after which they were grown on PDA for species
identification, using the morphological characteristics
described above (Isaac 1949, 1953). Microsclerotia of
some (later proven to be V. tricorpus) colonies were
sporulating, and those isolates could be put onto PDA
directly, by touching the conidiophore with a sterile
needle.
The identity of the ten soil isolates was checked with
species-specific primers (Robb et al. 1994). In brief,
DNA was extracted from conidia and mycelium and
purified, followed by a PCR using a primer pair specific
for amplification of either V. dahliae or V. tricorpus
rDNA of the ITS region. When amplification of the
DNA occurs, this is visible as a 334 bp band when the
PCR product is run through a gel.
Data on microsclerotial size were analysed using the
GLM procedure of SAS version 8.0 (SAS Institute,
Cary, NC). Specific research questions were tested with
t-tests (mentioned directly in the text) or using contrast
analyses (presented in the tables).
RESULTS
Experiment 1a. Morphology in the absence of soil
Verticillium dahliae on MSEA : Microsclerotia (n=150)
abundant (250–3000 per colony), shape usually
elongate to oval (Fig. 3), sometimes globose (A45 mc-1)
(Table 1), (30–)32–113(–190)r(10–)15–45(–55) mm
(the 5th and 95th percentiles between brackets) (average 62r29 mm ; length-width ratio (l/w) (1–)1.1–4.9
(–10.5), average 2.3), radially distributed through the
colonies (Fig. 1). Dark hyphae usually absent, though
1 isolate (A60 mc-1) exhibited dark hyphae connected
to some (1 %) of the microsclerotia or unconnected to
microsclerotia in several (4 %) of the colonies. Dark
mycelium throughout the colony absent. Yellow discoloration of the medium absent.
V. tricorpus on MSEA : Microsclerotia (n=90),
present only in isolates A36 mc-1 (1000–1500 per
colony), 227.84 mc-1 (1000–2500 per colony), and
in 24 % of the colonies of isolate 127.79A mc-1 (0–50
per colony), irregularly shaped (Fig. 4), (15–)25–85
J. C. Goud, A. J. Termorshuizen and W. Gams
1
825
2
500 µm
3
500 µm
4
50 µm
5
50 µm
6
500 µm
7
500 µm
8
50 µm
50 µm
Figs 1–8. Morphology of Verticillium dahliae and V. tricorpus on semi-selective media without soil. Fig. 1. V. dahliae colony on MSEA.
The microsclerotia are formed in a radial pattern, which is typical for V. dahliae, although a scattered pattern can also occur. Fig. 2. V. tricorpus
colony on MSEA, with dark mycelium in a radial pattern and microsclerotia in a scattered to intermediate between radial and scattered
pattern. Fig. 3. Globose to elongate V. dahliae microsclerotia on MSEA. Fig. 4. Globose to irregularly shaped V. tricorpus microsclerotia on
MSEA. Fig. 5. V. dahliae colony on EA, with microsclerotia in a scattered pattern. Fig. 6. V. tricorpus colony on EA, with microsclerotia
in a scattered pattern and dark mycelium in a radial pattern. Fig. 7. Large, globose to irregularly shaped V. dahliae microsclerotia with
connected dark hyphae on EA. Fig. 8. Globose to oval (to irregularly shaped, not shown) V. tricorpus microsclerotia on EA. Bars Figs 1–2
and 5–6=500 mm; Figs 3–4 and 7–8=50 mm.
Distinction of Verticillium dahliae and V. tricorpus
826
Table 2. Significance levelsa of type III sums of squares of overall effects in Experiment 1.
Variable
Factor
Length of
microsclerotia
Width of
microsclerotia
Visible surface of
microsclerotia
Speciesb
Mediumc
Soild
Speciesrmedium interaction
Speciesrsoil interaction
Mediumrsoil interaction
Speciesrmediumrsoil interaction
<0.0001
<0.0001
0.0006
0.0006
0.39
0.57
0.16
<0.0001
<0.0001
<0.0001
<0.0001
0.0048
<0.0001
0.22
0.0001
<0.0001
0.0001
<0.0001
0.26
0.0096
0.86
b
c
d
Calculated using the GLM procedure of SAS version 8.0.
Verticillium dahliae or V. tricorpus.
Modified soil extract agar or ethanol agar.
Present or absent.
(–270)r(10–)20–50(–70) mm (average 50r31 mm, l/w
=1.0–2.1(–5.4), average 1.7), usually scattered through
the colony, but sometimes intermediate between radial
and scattered (Fig. 2). Dark hyphae were present and
connected to all microsclerotia in every colony of isolates A36 mc-1 and 227.84 mc-1 (Fig. 4), but occurred
mostly separate from the microsclerotia in 52 % of the
colonies in isolate 127.79A mc-1. Dark hyphae that
were not connected to microsclerotia could not be
scored in colonies with abundant presence of dark
mycelium. Dark mycelium usually present and abundant (covering 80–100% of the colony area), but
absent in 127.79A mc-1 and scarce in A36 mc-1 (present
in 44 % of the colonies and on average covering 5 % of
the colony area) (Fig. 2). Yellow discoloration of the
medium present in all colonies of isolate 227.84 mc-1
and several colonies of isolate A36 mc-1, and absent in
all other colonies.
V. dahliae on EA : Microsclerotia (n=150) mostly
abundant (1000–6000 per colony), but variable in A63
mc-1 (several–2000 per colony), globose to elongate or
irregular in shape (Fig. 7), (30–)40–156(–220)r(25–)
30–90(–170) mm (average 86r56 mm, l/w=1.0–2.3(–4),
average 1.5), scattered through the medium (Fig. 5),
except for isolate A59 mc-1 which shows a radial distribution and isolate A63 mc-1 which shows a scattered
distribution in colonies containing few (<50) and a random distribution in colonies containing more microsclerotia. Dark hyphae connected or close to most
(80–95 %) of the microsclerotia in most isolates (Fig. 7),
but less frequent in A63 mc-1 (50 %) and A56 mc-1
(10 %), some dark hyphae also occurring unconnected
to microsclerotia. Dark mycelium throughout the colony mostly absent, but rare in A60 mc-1 and A63 mc-1,
covering 5% of the colony diameter. Yellow discoloration of the medium absent.
V. tricorpus on EA : Microsclerotia absent after 6 wk
of incubation. Dark mycelium abundant, covering
100 % of the colony diameter (Fig. 6). Dark hyphae
that were unconnected to microsclerotia could not be
scored properly, because of the dark mycelium. After
40
30
Width (µm)
a
20
10
30
40
50
60
Length (µm)
Fig. 9. Average length and width of microsclerotia of the ten
colonies from MSEA soil plates from agricultural fields (Experiment 2), together with the average length and width of
microsclerotia of Verticillium dahliae and V. tricorpus reference isolates (Experiment 1). $, V. dahliae field isolates; &,
V. tricorpus field isolates; #, average of V. dahliae reference
isolates ; %, average of V. tricorpus reference isolates ; the line
indicates a lengthrwidth of microsclerotia, i.e. the visible
microsclerotial surface, which was iteratively set at 1275 mm2.
scoring and leaving the Petri dishes at room temperature for another 6 wk, microsclerotia with dark hyphae
were formed in some colonies of isolate A36 mc-1
(0–200 per colony). Microsclerotia (n=30) globose
(Fig. 8) to irregular in shape, (20–)30–141(–160)r
20–70(–120) (average 62r39 mm, l/w=1.0–2.7(–3.2),
average 1.6), formed in a radial to scattered pattern
(Fig. 6). Yellow discoloration of the medium absent,
except for one colony of isolate 227.84 mc-1 and visible
only during the second week of incubation.
Experiment 1b. Morphology in the presence of soil
Verticillium dahliae on MSEA : Microsclerotia (n=150),
usually present (around 200 per colony ; less abundant
or sometimes absent in A59 mc-1 (0–100 per colony)),
J. C. Goud, A. J. Termorshuizen and W. Gams
globose to elongate, 20–80(–100)r10–50(–60) mm
(average 45r23 mm, l/w=1.0–4.8(–10.0), average 2.3),
radially distributed except for A59 mc-1 which showed
a scattered distribution and A45 mc-1 which showed
a scattered distribution in the colony centre and radial
distribution near the colony edges. Dark hyphae or
dark mycelium absent. No discoloration of the
medium.
V. tricorpus on MSEA : Microsclerotia (n=90) present in about half of the colonies (isolates A36mc-1 and
127.79A mc-1 always showed microsclerotia (10–100
per colony)), usually (around 90 %) with dark hyphae
connected to them, except for isolates 384.84 mc-1 and
Vtx mc-1 without, and 227.84 mc-1 with few microsclerotia (0–50 per colony), often irregularly shaped,
sometimes globose to oval, (10–)20–116(–260)r(10–)
15–50(–100) mm (average 48r28 mm, l/w=1.0–2.9
(–6.5), average 1.7), usually scattered through the colony, but sometimes intermediate between radial and
scattered. Dark mycelium present in about half of the
colonies, covering about 5 % of the total colony area.
Isolate 227.84 mc-1 showed numerous dark hyphae
that were not connected to microsclerotia. No discoloration of the medium.
V. dahliae on EA : Microsclerotia (n=90) present in
about half of the colonies, usually not abundant (0–100
per colony) except for isolate A45 mc-1 (around 400 per
colony), globose to elongate, (20–)30–140(–700)r(10–)
15–61(–120) mm (average 75r37 mm, l/w=(1.0–)1.2–
4.2(–14.0), average 2.2), scattered through the colony.
Dark hyphae or dark mycelium absent. No discoloration of the medium.
V. tricorpus on EA : Microsclerotia (n=90) present in
about half of the colonies, but never abundant (0–30
per colony), irregular in shape, small and elongate in
127.79A mc-1 and large and globose in A36 mc-1, (20–)
23–83(–170)r10–50(–80) mm (average 47r28 mm, l/w
=(1.0–)1.1–3.0(–5.3), average 1.8), usually scattered
through the colony, but radially distributed in 227.84
mc-1. Dark hyphae and dark mycelium present in
about half of the colonies, covering about 40% of the
colony area. No discoloration of the medium.
Experiment 1. Overall effects on microsclerotial size
Microsclerotial size is generally different for the two
species (larger for V. dahliae) and is severely affected by
choice of the medium (larger on EA) and presence or
absence of soil (larger if absent) (Table 2). The actual
size, the surface that is visible through a microscope,
is probably most clearly expressed in the factor
lengthrwidth. The significant mediumrsoil interaction for the factors width and visible surface (Table
2) illustrates that microsclerotial size is more stable on
MSEA. Significance levels of the speciesrsoil interaction and the three-way interaction are misleading,
because microsclerotia were often not formed.
The effect of adding soil on morphology : Presence of
a mixed soil microflora can alter the morphology of
827
the two species considerably. On MSEA, the presence
of soil resulted in shorter and narrower (P<0.001)
V. dahliae microsclerotia, but had no significant effect
on the size of V. tricorpus microsclerotia. On EA, the
presence of soil resulted in shorter (P=0.036) and
narrower (P<0.001) V. dahliae microsclerotia, whereas
in V. tricorpus, soil induced formation of microsclerotia
in two isolates, and accelerated formation of microsclerotia in isolate A36 mc-1, without affecting the size.
On MSEA without soil, V. dahliae microsclerotia
were longer (P=0.037) but not wider than those of
V. tricorpus, whereas in the presence of soil, V. tricorpus microsclerotia were wider (P=0.015) and
slightly, but not significantly, longer than those of V.
dahliae (Fig. 9). On EA, V. dahliae microsclerotia were
longer and wider than those of V. tricorpus, both
without and with soil (P<0.004). Despite these consistent differences, EA is less suited for quantification
of V. dahliae in soil, because V. dahliae microsclerotia
are not always formed (see above). Morphological
characteristics suitable to separate V. dahliae and
V. tricorpus on MSEA in the presence of soil are summarised in Table 3.
Experiment 2. Classification of colonies of soil
samples from agricultural fields
On the basis of the differential features recognised on
MSEA with added soil (Experiment 1b), five of ten field
soil colonies were scored as Verticillium dahliae and five
as V. tricorpus (Table 3). In colony no. 2, microsclerotia
were oval to elongate and arranged in a radial pattern,
classifying it as V. dahliae. Colony no. 6 had globose to
elongate microsclerotia in a more irregular pattern, and
was also classified as V. dahliae, based on the elongate
shape of the microsclerotia. Colonies nos 1, 3 and 7 had
dark hyphae attached to the microsclerotia, classifying
them as V. tricorpus. This identification was confirmed
for colonies nos 1 and 3, which contained irregularly
shaped microsclerotia. A comparison of the visible
surface (lengthrwidth) of the microsclerotia of each
field soil colony against the reference isolates of V.
dahliae and V. tricorpus (Experiment 1) confirmed these
identifications, and identified three more V. dahliae and
two more V. tricorpus isolates (Table 3). Checking the
identity of the purified isolate by PCR with specific
primers and morphology on PDA confirmed the original identification based on morphological characteristics in the MSEA soil plates (Table 3).
DISCUSSION
Morphology of Verticillium species can be highly variable on different semi-selective agar media. Shape of
microsclerotia and abundance of dark hyphae of
V. dahliae on EA resembles morphology of V. tricorpus
on MSEA. Moreover, the distribution of microsclerotia
through the colony on EA is scattered, whereas on
Isolate(s)
Colony
patterna
Microsclerotial
shape
Approx. % of
microsclerotia
with connected
dark hyphae
V. tricorpus
Species identification
of isolates based
on morphology
on MSEA in
the presence of
soil
PCR
specific
primersc
Morphology
on PDAd
NAe
NA
NA
V. dahliae
V. dahliae
Probabilityb based on
microsclerotial size of
the field isolates being
Dark
mycelium
Average
microsclerotial
surface
(lengthrwidth)
(mm2)
V. dahliae
Absent
1190
Check of identity of purified
isolate by
Reference isolates (Exp. 1b)
V. dahliae
Radial,
scattered or
intermediate
V. tricorpus
Scattered or
intermediate
Globose, oval to
elongate
0
Globose, oval to
irregular
90
Absent or
present
1725
NA
NA
NA
V. tricorpus
V. tricorpus
Field isolates (Exp. 2)
1
Intermediate
2
Radial
3
Scattered
4
Scattered
5
Intermediate
6
Intermediate
7
Intermediate
8
Scattered
9
Intermediate
10
Scattered
Irregular
Oval-elongate
Globose-irregular
Globose-oval
Globose-oval
Globose-elongate
Globose-oval
Globose-oval
Globose-oval
Globose-oval
50
0
80
0
0
0
70
0
0
0
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
Absent
1690
720
1520
2240
1020
880
1710
1810
1030
1260
0.24f
0.26
0.43
0.01
0.69
0.46
0.22
0.14
0.70
0.87
0.94f
0.02
0.66
0.23
0.10
0.05
0.97
0.84
0.11
0.28
V. tricorpus
V. dahliae
V. tricorpus
V. tricorpus
V. dahliae
V. dahliae
V. tricorpus
V. tricorpus
V. dahliae
V. dahliae
V. tricorpus
V. dahliae
V. tricorpus
V. tricorpus
V. dahliae
V. dahliae
?h
V. tricorpus
V. dahliae
V. dahliae
V. tricorpus
V. dahliae
V. tricorpusg
V. tricorpus
V. dahliae
V. dahliae
V. tricorpusg
V. tricorpus
V. dahliae
V. dahliae
Distinction of Verticillium dahliae and V. tricorpus
Table 3. Morphological characteristics distinguishing Verticillium dahliae and V. tricorpus on MSEA in the presence of soil.
a
Colony pattern is unclear when few microsclerotia are present.
Contrast analysis of each isolate of Experiment 2 against the average of all isolates of V. dahliae and all microsclerotia forming isolates of V. tricorpus, respectively, of Experiment 1b on MSEA in the
presence of soil (GLM procedure, SAS version 8.0, SAS, Cary, NC).
c
Robb et al. (1994).
d
Isaac (1949, 1953).
e
Not applicable.
f
The higher probability value (either V. dahliae or V. tricorpus) indicates the most likely possibility.
g
These isolates caused yellow coloration of the PDA medium.
h
No signal with either V. dahliae or V. tricorpus primers.
b
828
J. C. Goud, A. J. Termorshuizen and W. Gams
MSEA it is usually (but not always) radial. This is the
first report of the occurrence of dark hyphae of V.
dahliae in vitro. In vivo production of dark hyphae has
sometimes been reported in cotton (Garber & Houston
1966) and olive (Rodrı́guez-Jurado 1993). Researchers
only familiar with V. dahliae on pectate-based agars
can easily be confused when occasionally working
with EA.
On MSEA, microsclerotia are the most important
structures to recognise Verticillium colonies in the
presence of soil, because many other fungi present in
the soil are able to form melanised mycelium or chlamydospores (Domsch et al. 1980). Therefore, presence
of dark hyphae is a useful characteristic only, when
the dark hyphae are connected to the microsclerotia.
Because this is the case with most of the V. tricorpus
microsclerotia and never occurs in V. dahliae, this
characteristic is very important for discrimination, and
easy to observe. Shape of the microsclerotia is a useful
characteristic, when the colony contains elongate
(V. dahliae) or irregularly shaped (V. tricorpus) microsclerotia, which is also easy to observe. The pattern in
which microsclerotia are formed discriminates only
when it is clearly radial. When microsclerotia are
scarce, the pattern is often unclear. Dark mycelium,
when present, identifies a colony as V. tricorpus. However, dark mycelium did not occur in any of the V. tricorpus field colonies tested, so this characteristic does
not seem to be useful for practice.
All previously mentioned qualitative characteristics
fail to discriminate between V. dahliae and V. tricorpus
when dark hyphae and dark mycelium are absent, and
microsclerotia are globose to oval and arranged in an
irregular pattern. Size of the microsclerotia is a useful
character for discrimination, despite the overlap in
length and width of the field isolates with the reference
isolates (Fig. 9). Size, however, is more clearly expressed as the average lengthrwidth of the microsclerotia, i.e. the visible surface. The visible surface
differentiates clearly to the experienced observer, even
without measuring the microsclerotia on a standard
basis. Using both qualitative and quantitative characteristics, it is possible to discriminate V. dahliae and
V. tricorpus on MSEA in the presence of soil.
These observations provide a more practical (yet
reliable) method to characterise Verticillium colonies
829
on MSEA soil dilution plates. The alternatives, to
purify individual colonies and identify them using
morphological or molecular characteristics, are too
laborious and costly if large numbers of colonies need
to be identified.
These observations in part confirm those of Davis &
McDole (1979) and Davis et al. (2000) on the pectate
based agar NPX (Butterfield & DeVay 1977) and of
Huisman (1988) on a similar pectate medium. All
authors mentioned the radially grouped V. dahliae, and
scattered, dark hyphae bearing V. tricorpus microsclerotia, as well as the microsclerotial size difference.
However, they did not mention any deviations from
these typical characteristics.
MSEA is used for quantification of V. dahliae in
research (Termorshuizen et al. 1998) and commercially
(Anon. 1997) in soils with the two species present.
Quantitative detection using MSEA has given a good
prediction of disease for V. dahliae, e.g. in Acer platanoides (Goud et al. 2001), but for V. tricorpus the
method was not conclusive because some isolates
did not form microsclerotia. EA cannot be used for
quantification of V. dahliae in soil. EA is suitable
for working in the absence of soil, e.g. during plating
of stem pieces of crops that can suffer from both
species, like potato (e.g. Nagtzaam, Termorshuizen &
Bollen 1997).
The addition of soil to MSEA Petri dishes affected
microsclerotial size in V. dahliae more than in V. tricorpus isolates, which could be explained by the nature
of the two species. V. dahliae is regarded as a plant
pathogen, with low saprobic ability, whereas V. tricorpus is regarded as being more a soil inhabitant (Isaac
1967). The formation of microsclerotia by V. tricorpus
on EA in the presence of soil but not in pure culture, or
only after prolonged incubation, might be induced by
competing microorganisms. Probably, microsclerotia
are more durable resting structures than melanised
mycelium, and are more frequently formed in the
presence of other soil organisms. This hypothesis needs
further testing.
In conclusion, the morphological characters used to
identify pure cultures of dark Verticillium species cannot be used uncritically for soil dilution plates. For
distinguishing V. dahliae and V. tricorpus on MSEA
soil dilution plates we recommend the following key:
Key to distinguish Verticillium dahliae and V. tricorpus
1
2(1)
3(2)
4(3)
5(4)
Dark hyphae or dark mycelium present .
.
.
.
.
.
.
.
.
Dark hyphae and dark mycelium absent
.
.
.
.
.
.
.
.
Microsclerotia irregularly shaped
.
.
.
.
.
.
.
.
.
.
Microsclerotia not irregularly shaped ; globose to oval to elongate
.
.
.
.
Microsclerotia elongate
.
.
.
.
.
.
.
.
.
.
.
Microsclerotia globose to oval
.
.
.
.
.
.
.
.
.
.
Distribution of microsclerotia within medium clearly radial
.
.
.
.
.
Distribution of microsclerotia within medium not clearly radial
.
.
.
.
Average surface (average lengthraverage width) of microsclerotia (n=10)<1275 mm2
Average surface (average lengthraverage width) of microsclerotia (n=10)>1275 mm2
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
tricorpus
.
2
tricorpus
.
3
dahliae
.
4
dahliae
.
5
dahliae
tricorpus
Distinction of Verticillium dahliae and V. tricorpus
ACKNOWLEDGEMENTS
The authors wish to thank H. W. ‘Bud ’ Platt and George S. Mahuku
(Agriculture and Agri-Food Canada, Research Centre, Charlottetown, Prince Edward Island) for supervision of the molecular analysis, J. Wil Veenbaas and Hennie A. van Kesteren (Plant Protection
Service, Wageningen) for provision of isolates, Rien Hooftman
(Inspection Service for Horticulture, Roelofarendsveen) for supply of
soil dilution plates containing colonies of Verticillium of doubtful
identity, and Ariena H. C. van Bruggen (Biological Farming Systems,
Wageningen University) for helpful comments on the manuscript.
This work was supported by the Product Board for Horticulture in
the Netherlands and by the European Commission Framework Programme 5; project reference number QLRT-1999-1523 (Verticillium
in trees).
REFERENCES
Anon. (1997) [Verticillium threat for growing road-side trees.] De
NAKB krant Laboratorium special : 3. [In Dutch.]
Butterfield, E. J. & DeVay, J. E. (1977) Reassessment of soil assays
for Verticillium dahliae. Phytopathology 67: 1073–1078.
Davis, J. R., Everson, D. O., Sorensen, L. H. & Schneider, A. T.
(2000) Association of Verticillium tricorpus with soil suppressiveness of verticillium wilt of potato. In Advances in Verticillium.
Research and Disease Management (E. C. Tjamos, R. C. Rowe,
J. B. Heale & D. R. Fravel, eds): 347–351. American Phytopathological Society Press, St Paul, MN.
Davis, J. R. & McDole, R. E. (1979) Influence of cropping sequences
on soil-borne populations of Verticillium dahliae and Rhizoctonia
solani. In Soil Borne Plant Pathogens (B. Schippers & W. Gams,
eds): 399– 405. Academic Press, London.
DeVay, J. E., Forrester, L. L., Garber, R. H. & Butterfield, E. J.
(1974) Characteristics and concentration of propagules of Verticillium dahliae in air-dried field soils in relation to the prevalence of
verticillium wilt in cotton. Phytopathology 64: 22–29.
DeVay, J. E., Garber, R. H., Wakeman, R. J. & Paplomatas, E. J.
(1988) Seed and soil treatments for control of seed and seedling
diseases of cotton in California. Proceedings of the Beltwide Cotton
Production Research Conferences, USA: 23–26.
Domsch, K. H., Gams, W. & Anderson, T. H. (1980) Compendium of
Soil Fungi. Vol. 1. Academic Press, London.
Garber, R. H. & Houston, B. R. (1966) Penetration and development
of Verticillium albo-atrum in the cotton plant. Phytopathology 56:
1121–1126.
Goud, J. C., Blok, W. J., Coenen, G. C. M., Lans, T. & Termorshuizen, A. J. (2001) Effect of biological soil disinfestation on
inoculum levels of Verticillium dahliae and inoculum density – disease incidence relationships in Norway maple and Southern
Catalpa (Abstract). In Programme and Book of Abstracts of the
8th International Verticillium Symposium, Córdoba, Spain, 5–8
November, 2001: 98.
Harris, D. C., Yang, Y. R. & Ridout, M. S. (1993) The detection and
estimation of Verticillium dahliae in naturally infested soil. Plant
Pathology 42: 238–250.
Hiemstra, J. A. (1998) Some general features of verticillium wilts in
trees. In A Compendium of Verticillium Wilts in Tree Species (J. A.
Hiemstra & D. C. Harris, eds): 5–11. CPRO-DLO, Wageningen.
830
Huisman, O. C. (1988) Seasonal colonization of roots of field-grown
cotton by Verticillium dahliae and V. tricorpus. Phytopathology 78:
708–716.
Isaac, I. (1949) A comparative study of pathogenic isolates of Verticillium. Transactions of the British Mycological Society 32:
137–159.
Isaac, I. (1953) A further comparative study of pathogenic
isolates of Verticillium: V. nubilum Pethybr. and V. tricorpus
sp. nov. Transactions of the British Mycological Society 36:
180–195.
Isaac, I. (1967) Speciation in Verticillium. Annual Review of Phytopathology 5: 201–222.
Karapapa, V. K., Bainbridge, B. W. & Heale, J. B. (1997) Morphological and molecular characterization of Verticillium longisporum comb. nov., pathogenic to oilseed rape. Mycological Research 101: 1281–1294.
Messner, R., Schweigkofler, W., Ibl, M., Berg, G. & Prillinger, H.
(1996) Molecular characterization of the plant pathogen Verticillium dahliae Kleb. using RAPD-PCR and sequencing of the 18S
rRNA gene. Journal of Phytopathology 144: 347–354.
Nadakavukaren, M. J. & Horner, C. E. (1959) An alcohol agar
medium selective for determining Verticillium microsclerotia in
soil. Phytopathology 49 : 527–528.
Nagtzaam, M. P. M., Termorshuizen, A. J. & Bollen, G. J. (1997)
The relationship between soil inoculum density and plant infection
as a basis for a quantitative bioassay of Verticillium dahliae.
European Journal of Plant Pathology 103: 597–605.
Paplomatas, E. J., Tzalavaras, S. & DeVay, J. E. (2000) Use of
Verticillium tricorpus as a biocontrol agent of Rhizoctonia solani
on cotton seedlings. In Advances in Verticillium: research and
disease management (E. C. Tjamos, R. C. Rowe, J. B. Heale &
D. R. Fravel, eds): 253–256. American Phytopathological Society
Press, St Paul, MN.
Pegg, G. F. & Brady, B. L. (2002) Verticillium Wilts. CABI Publishing, Wallingford.
Robb, E. J., Hu, X., Platt, H. W. & Nazar, R. N. (1994) PCR assays
for the detection and quantification of Verticillium albo-atrum, V.
dahliae and V. tricorpus in potato. In Modern Detection Assays for
Plant Pathogenic Fungi (M. Dewey, R. Oliver & A. Schots, eds):
83–90. CAB International, Wallingford.
Rodrı́guez-Jurado, D. (1993) [Host–parasite interactions in the
wilt of olive (Olea europaea L.) induced by Verticillium dahliae
Kleb.] PhD. thesis, University of Córdoba, Córdoba, Spain.
[In Spanish.]
Skotland, C. B. (1971) Pathogenic and nonpathogenic Verticillium
species from South Central Washington. Phytopathology 61:
435– 436.
Smith, H. C. (1965) The morphology of Verticillium albo-atrum, V.
dahliae and V. tricorpus. New Zealand Journal of Agricultural
Research 8: 450– 478.
Termorshuizen, A. J., Davis, J. R., Harris, D. C., Huisman, O. C.,
Gort, G., Lazarovits, G., Locke, T., Melero Vara, J. M., Mol, L.,
Paplomatas, E. J., Platt, H. W., Powelson, M., Rouse, D. I., Rowe,
R. C. & Tsror, L. (1998) Interlaboratory comparison of methods to
quantify microsclerotia of Verticillium dahliae in soil. Applied and
Environmental Microbiology 64: 3846–3853.
Corresponding Editor: D. L. Hawksworth